Rotenoids and Isoflavones from Xeroderris stuhlmannii (Taub.) Mendonça & E.P. Souza and Their Biological Activities

The phytochemical study of the ethanolic extract of the leaf of Xeroderris stuhlmannii led to the isolation of five hitherto unreported compounds including two isoflavones (1–2), and three rotenoids (3–5), along with eight known isoflavonoid derivatives (6–13) and one pterocarpan derivative (14). The structures of the new compounds and those of the known ones were established by the spectroscopic (1D and 2D NMR) and spectrometric (HRESIMS) techniques as well as a comparison of their spectroscopic data with those reported in the literature. The leaf extract, fractions, and isolated compounds were tested for their antibacterial effects against nine bacterial strains. Compounds 3, 8, 11, and 12 showed a significant antibacterial effect, with a minimum inhibitory concentration (MIC) value of 62.5 µg/mL each, against Salmonella typhi, Staphylococcus aureus, Klessiella pneumonae, and Escherichia coli, respectively. In addition, the leaf extract, fractions, and isolated compounds were tested for their antifungal effects against four fungal strains. The hexane fraction showed a significant antifungal effect with an MIC value of 125 µg/mL against Candida parasilosis, whereas compounds 3, 8, and 12 showed significant antifungal activity with an MIC value of 62.5 µg/mL, each against Candida parasilosis, Candida albicans, and Candida krusei, respectively.


Introduction
Xeroderris genus belongs to the Fabaceae family, commonly known as Leguminoseae, and is an extremely rich source of biologically active compounds mainly flavonoid derivatives, which is the major class of secondary metabolites found in the family [1,2]. These bioactive phytochemicals possess antibacterial, antioxidant, antifungal, and antimalarial activities [3][4][5]. Despite the existing various solutions for health care, drug resistance is common. Thus, the research of new compounds to tackle biological resistance is urgent [6]. Based on the various diversity of secondary metabolites from the Fabaceae family [3] and the ethnopharmacological report on Xeroderris stuhlmannii (Taub) Mendonça and Souza, we examined the leaves of X. stuhlmannii, one of the two species of Xeroderris genus [7]. X. stuhlmannii grows in the West Region of Cameroon and is also called "wing pod" or "wing bean" in English and "mumundu" in the South-West region of Cameroon [8,9]. X. stuhlmannii is a tree, growing up to 18-27 m tall and 120 cm in diameter. Its leaves are alternate at the tips of the branches and are 6-12 mm long, while fruits come in the form X. stuhlmannii is a tree, growing up to 18-27 m tall and 120 cm in diameter. Its leaves are alternate at the tips of the branches and are 6-12 mm long, while fruits come in the form of a linear-oblong, globous, and contain one or two bean-shaped seeds [10]. The leaves of X. stuhlmannii are used in traditional folk medicine to treat colds and stomach pains, while the boiled roots are used to fight against malaria [4,9]. LC-MS analysis of the bark extract of X. stuhlmannii proposed various classes of secondary metabolites such as rotenoids, flavonoids, polyphenols, tri-terpenoids, steroids, and other phytochemicals. Moreover, bark extract indicated antibacterial activity on Salmonella appendicitis, Coliform, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherishia coli [5]. We report, herein, the isolation and structure determination of isoflavones and rotenoids derivatives from the ethanolic leaf extract of X. stuhlmannii. In addition, antibacterial, antifungal, and antioxidant activities of leaf extract, fractions, and some isolated compounds were also evaluated.
The ABX system was easily located on the A-ring, due to the deshielded value of H-8 induced by the anisotropic effect of the carbonyl C-4. Moreover, the AA'BB' spin coupled system appearing at δ H 7.52 (2H, dd, J = 8.8 Hz, H-2 /6 ) and δ H 6.99 (2H, dd, J = 8.8 Hz, H-3 /5 ) indicated a para-substituted B-ring. Furthermore, the 1 H NMR spectrum displayed singlet protons at δ H 3.86 (3H, s) for the O-Me group, while a side-chain moiety was shown by 13 C (Table 1) and DEPT data analysis to contain 10 carbons and one hydroxy-group, suggesting the presence of either a geraniol or nerol moiety [22]. Fuendjiep et al. showed that 13 C data, particularly the chemical shift of the methyl at the CH 3 -9" and that of the methylene C-4", aid to distinguish a geranyl side chain [12]. The chemical shift at δ C 16.8 and 42.2 ppm observed for methyl and methylene, respectively, combined with the biogenetic consideration confirmed the presence of a geraniol side chain in compound 2 [12]. The positions of these two units were determined using the HMBC spectrum. In fact, the correlation between the methoxy protons at δ H 3.86 and the aromatic protons H-2 /6 with carbon C-4 at δc 159.6 allowed us to locate the methoxy group at C-4 , and otherwise, the cross-peaks observed between H-8 (δ H 8.22), the oxymethylenic protons H-1" (δ H 4.66) and carbon C-7 (δc 163.2) highlighted that the geranyloxy moiety was located at C-7. On the basis of the above spectroscopic evidence, the structure of 2 was deduced to be 7-(((2E,5E)-7hydroxy-3,7-dimethylocta-2,5-dien-1-yl)oxy)-3-(4-methoxyphenyl)-4H-chromen-4-one and trivially named Stuhlmannione B.

Antibacterial Evaluation
The antibacterial effects of the EtOH leaf extract, hexane, and AcOEt fractions as well as all the isolated compounds were evaluated against ATCC strains using the microtiter broth dilution method to determine the MIC and MBC (minimal inhibitory and bactericidal concentration) [26]. The results (Table 3)

Antifungal Evaluation
The antifungal activity of the EtOH leaf extract, hexane, and AcOEt fractions as well as all the isolated compounds were evaluated against the four fungal strains Candida albicans, Candida krusei, Candida parasilosis, and Cryptococcus neoformans following a standard protocol. Table 4 shows the antifungal activity of leaf extract, fractions, and some isolated compounds. The leaf extract was found to be inactive against all the tested strains, whereas the hexane fraction evoked moderate activity against C. parasilosis with the MIC value of 250 µg/mL and a fungicidal effect with a ratio MFC/MIC of 2. Compound 12 highlighted moderate activity against C. krusei with an MIC value of 62.5 µg/mL, weak activity against C. parasilosis and C. neoformans with MIC values of 125 µg/mL, and fungicidal effects with MFC/MIC ratios of 4, 2, and 2, respectively. Compound 3 showed moderate activity against C. parasilosis with an MIC value of 62.5 µg/mL, weak activity against C. albicans and C. neoformans with MIC values of 125 µg/mL, and fungicidal effects with MFC/MIC ratios of 2, 4, and 2, respectively. Compound 6 revealed weak activity against C. parasilosis and C. neoformans with MIC values of 125 µg/mL and fungicidal effects with MFC/MIC ratio of 2 each. Compound 8 was evaluated with moderate activity against C. albicans with an MIC value of 62.5 µg/mL, weak activity against C. krusei with an MIC value of 125 µg/mL, and fungicidal effects with MFC/MIC ratios of 4 and 2, respectively. The other compounds were found to be either weakly active or inactive on the tested strains.

Antioxidant Evaluation
It has been demonstrated that more than one method is necessary to elucidate the antioxidant capacity of samples because these assays differ in the principles and experimental conditions. In this study, the antioxidant activity of the ethanolic leaf extract, hexane, and AcOEt fractions as well as the isolated compounds were tested using the radical scavenging activities and reducing power as listed in Table 5. The ethanolic leaf extract showed significant scavenging activity against 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) with RS a50 of 13.44 ± 0.82 and 2,2-dipheny-1-picrylhydrazyl (DPPH) with RS a50 of 14.97 ± 0.86 µg/mL, while a poor reducing power was observed within the ferric reducing antioxidant power (FRAP) assay. These results suggest that the ethanol leaf extract contains compounds that could act as a free radical scavenger that is, capable of donating hydrogen or electron to a free radical in order to stabilize the odd electron which is responsible for radical's reactivity [27]. The fractions and isolated compounds were found to be either weakly active or inactive. The absence of activity in the isolated compounds may be due to the lack of proton donors in almost all their structures. Although the known compounds displayed either weak or no activity in this work, griffonianone D (7) was reported to possess anti-inflammatory effects in different experimental models of inflammation [11], while ichthynone (9) displayed a weak cytotoxic effect on cancer cells. [28]. Formonotin (10) was reported to exhibit anticancer activity [29], whereas maximaisoflavone B (13) and abrusprecatin (14) exhibit weak antiplasmodial activities [19,30]. To the best of our knowledge, no biological activity is reported for odoratin (6), conrauinone (8), 7-O-geranylformononetin (11), and conrauinone C (12).

General Experimental Procedures
HR-ESI were generated on either an Agilent 6220 TOF LCMS mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with perfluorokerosene as reference substance or Synapt G2-S (Waters, Manchester, UK, Details in the Supplementary Materials) for ESI-HR-MS. The NMR spectra were recorded at 500 MHz for 1 H and 125 MHz for 13 C, on Bruker DRX 500 or Avance NEO spectrometer with a cryo probe CTCI ( 1 H/ 13 C/ 15 N) (Bruker, Rheinstetten, Germany) in CDCl 3 , C 3 D 6 O, and DMSO-d 6 . All the chemical shifts are given in δ (ppm) with reference tetramethylsilane (TMS) (Sigma-Aldrich, Munich, Germany) to the residual solvent signal, while coupling constants (J) were measured in Hz. Data processing and evaluation were performed using the Topspin Software Version 4.0.7 (Bruker, Rheinstetten, Germany). For the determination of the specific optical rotation, the polarimeter Jasco P-2000 (Jasco, Groß-Umstadt, Germany) digital polarimeter with a 5-cm cuvette was employed. The aperture was set to 1.8 mm. Column chromatography was performed using Sephadex LH-20 gel (Amersham Pharmacia Biotech, Uppsala, Sweden) and silica gel 60 (0.040-0.063 mm, Merck, Darmstadt, Germany). Preparative HPLC was performed on a Jasco System (Jasco, Groß-Umstadt, Germany, see details the Supplementary Materials). Thin layer chromatography (TLC) was carried out on silica gel 60 F 254 (Merck, Darmstadt, Germany) plates developed with hexane-EtOAc, EtOAc, and EtOAc-MeOH. While spots were detected by spraying with 10% H 2 SO 4 reagent followed by heating. The molecular composition of the isolated compounds was identified by accurate mass determinations. All reagents used were of analytical grade.

Plant Material
The leaves of X. stuhlmannii were collected in Tonga, west region of Cameroon, in November 2020. The plant was identified by Tacham Walter of National Herbarium of the Cameroon, where a voucher specimen (No. 6011/SRF/CAM) has been deposited.

Antibacterial Activity
The antibacterial activity was implemented against nine strains (Pseudomonas aeruginosa (ATCC01); Staphylococcus aureus (ATCC25922); Escherishia coli (ATCC10536); Klessiella pneumonae (ATCC13883); Shigella flexineri; Shigella dysenteria; Salmonella typhimurium; Salmonella typhi (ATCC6539), and Salmonella enteritidis). The broth microdilution method was used for susceptibility testing of bacteria species in 96-well microtiter sterile plates as previously described [26,31,32]. Briefly, the crude extracts were dissolved in 5% DMSO solution and diluted with Mueller Hinton broth to obtain a stock concentration of 2000 µg/mL for the extracts, 1000 µg/mL for fractions, and 500 µg/mL for the isolated compounds. This gave a concentration range of 1000-0.96 µg/mL, 500-0.96 µg/mL, and 250-0.96 µg/mL respectively. One hundred microliters of each bacterial suspension (containing about 1.5 × 10 6 CFU/mL) was added, respectively, to the wells containing the test samples and mixed thoroughly to give final concentrations ranging from 500 to 0.48 µg/mL for extract, 250 to 0.48 µg/mL for fraction, and 125 to 0.48 µg/mL for isolated compounds. Ciprofloxacin ® (Bayer, Leverkusen, Germany) at concentration of 125-0.48 µg/mL was used as the standard reference. The assay microtiter plates were incubated at 37 • C for 24 h. Inhibitory concentrations of the extracts were detected after addition of 50 µL to 0.2 mg/mL p-iodonitrotetrazolium chloride (INT) (Sigma-Aldrich, Johannesburg, South Africa) and incubated at 37 • C for 30 min. These preparations were further incubated at 37 • C for 48 hrs, and bacterial growth was revealed by the addition of INT as mentioned above. The smallest concentration at which no color change was observed was considered as the MBC. The tests were performed in duplicates. The ratio MBC/MIC was calculated to determine the bactericidal (MBC/MIC ≤ 4) and bacteriostatic (MBC/MIC > 4) effects.

Antifungal Activity
The inocula of yeasts were prepared from 48 h old cultures by picking numerous colonies and suspending them in sterile saline (NaCl) solution (0.9%). Absorbance was read at 530 nm and adjusted with the saline solution to match that of a 0.5 McFarland standard solution, corresponding to about 10 6 yeast cells/mL (CLSI, CLSI, formerly national committee for clinical and laboratory standards, NCCLS, 2008). MIC of each extract was determined by using broth microdilution techniques according to the guidelines of CLSIfor yeasts (M27-A2). Stock solutions of the test extracts were prepared in 5% aqueous DMSO solution and diluted with sabouraud dextrose broth (SDB) to give a concentration of 1 mg/mL. This was serially diluted two-fold to obtain a concentration range of 500-0.24 µg/mL for extracts and 125-0.24 µg/mL for compounds. The final concentration of DMSO in the well was less than 1% (preliminary analysis with 1% DMSO did not inhibit the growth of the test organisms). The plates were covered with a sterile lid and incubated on the shaker at 37 • C for 48 h (for yeasts) or at 28 • C for 7 days (for dermatophytes) [33][34][35]. MICs were assessed visually after the corresponding incubation period and were taken as the lowest product concentration at which there was no or virtually no growth. The assay was repeated three times. Nystatin (for yeasts) and griseofulvin (for dermatophytes) were used as positive controls.

Antioxidant Activity
• DPPH radical scavenging assay The free radical scavenging activities of the sample were evaluated using the DPPH analysis as described by Noghogne et al. [36]. The radical scavenging activities of crude extract were evaluated through spectrophotometer using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. When DPPH reacts with an antioxidant sample, which can donate hydrogen, it is reduced. The changes in color were measured at wave length 517 nm under UV/visible light spectrophotometer (Infinite M200, TECAN, Männedorf, Switzerland). The extract (1000 µg/mL) was two-fold serially diluted with methanol. Fifty microliters of the diluted extract (1000 µg/mL) in methanol were mixed with 150 µL of 0.02% of 2,2-diphenyl-1-picrylhydrazyl (DPPH) methanol solution, giving a final extract concentration range from 250 to 1.9531 µg/mL (250, 125, 62.5, 31.25, 15.625, 7.8125, 3.9062, and 1.9531 µg/mL). After 30 min of incubation in the dark at room temperature, the optical density was measured. Ascorbic acid (Vitamin C) was used as positive control. Each assay was done in triplicate, and the results recorded as the mean ± standard deviation (SD). The radical scavenging activity (RSA, %) was calculated as follows: Absorbance of DPPH -Absorbance of sample Absorbance of DPPH × 100 (1) • ABTS radical scavenging assay The radical scavenging activities of the samples were evaluated spectrophotometrically using the 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulphonic) acid (ABTS) free radical [37]. When ABTS reacts with an antioxidant compound, which can donate hydrogen, it is reduced. The changes in color were measured at 734 nm under UV/visible light spectrophotometer (Infinite M200 (TECAN, Männedorf, Switzerland). Pure methanol was used to calibrate the counter. The extract (1000 µg/mL) was twofold serially diluted with methanol. Twenty-five microliters of the diluted extract were mixed with seventy-five µL of 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulphonic) acid (ABTS) methanol solution, to give a final extract concentration range of 250-1.9531 µg/mL (250, 125, 62.5, 31.25, 15.625, 7.8125, 3.9062, and 1.9531 µg/mL). After 30 min of incubation in the dark at room temperature, the optical densities were measured at 734 nm. Ascorbic acid (Vitamin C) was used as a control. Each assay was done in triplicate, and the results, recorded as the mean ± standard deviation (SD) of the three findings, were presented in tabular form. The radical scavenging activity (RSA, in %) was calculated as follows: Absorbance of ABTS -Absorbance of sample Absorbance of ABTS × 100 (2) • FRAP assay The ferric reduction potential (conversion potential of Fe 3+ to Fe 2+ ) of the samples was determined according to the method described by Padmaja et al. [38]. Briefly, the samples were first dissolved as for the DPPH assay. 25 µL from each dilution was introduced into a new microplate, and 25 µL of 1.2 mg/mL Fe 3+ solution was added. The plates were pre-incubated for 15 min at ambient temperature. After this time, 50 µL of 0.2% orthophenanthroline was added to obtain final extract concentrations of 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625, and 1.95325 µg/mL. The reaction mixtures were further incubated for 15 min at ambient temperature after which the absorbance was measured at 505 nm under UV/visible light spectrophotometer (Infinite M200 TECAN, Männedorf, Switzerland) against the blank (made of 25 µL methanol + 25 µL Fe 3+ + 50 µL ortho-phenanthroline). Ascorbic acid (Vitamin C) was used as a positive control. The assay was performed in triplicate. From the obtained OD (optical density), reducing percentages were calculated for each concentration and used to determine the RC 50 from dose-response curves.

Conclusions
In summary, we have conducted the successful isolation of 15 compounds, including two new isoflavone derivatives (1-2) and three new rotenoid derivatives (3)(4)(5), together with nine known compounds (6-14) from X. stuhlmannii. The biological evaluations revealed that the leaf extract showed a general trend to exhibit weaker antibacterial and antifungal activities than the hexane fraction. From the evident results, compounds 3, 12, and 8 highlighted the greatest antibacterial and antifungal activities. However, no antioxidant activity was observed with the isolated compounds. From these results, further study may be done to confirm the use of the leaves in traditional folk medicine to treat diarrhoea, coccidiosis, endoparasites, fungal infections, and lethargic birds.